title: Impact of monocytic cells on recovery of uncultivable bacteria from atherosclerotic lesions.
abstract: OBJECTIVE
  Epidemiological evidence suggests that infections may contribute to atherogenesis. However, with the exception of Chlamydophila pneumoniae, cultivable bacteria have not been recovered from atherosclerotic lesions. Therefore, we aimed at developing an approach to recover uncultivable bacteria from atherectomy tissues.
  
  
  METHODS
  We cultured homogenates from atherectomy specimens from seven nonseptic patients undergoing surgery for arterial obstruction either alone or together with THP-1 monocyte-like cells. We performed 16S rDNA analysis, biochemical tests, random amplification of polymorphic DNA PCR analysis, quantitative polymerase chain reaction (qPCR) and immunohistofluorescence to identify the cultivated bacteria. Wilcoxon signed-rank tests were used to determine whether THP-1 treatment yielded a higher number of isolates than did the untreated controls.
  
  
  RESULTS
  We recovered more bacteria from cocultures of atherectomy specimens with THP-1 cells than atherectomy specimens cultured alone. On average, tissue homogenates incubated with THP-1 cells versus control yielded 124 vs. 22 colony-forming units, a median of 140 vs. 7, respectively (P = 0.02). We recovered 872 isolates of limited number of species, including Propionibacterium acnes, Staphylococcus epidermidis and Streptococcus infantis and the fastidious anaerobe Porphyromonas gingivalis, and confirmed its presence in tissue using double immunofluorescence imaging. qPCR demonstrated the presence of ≥3.5 × 10(3) P. gingivalis genomes per gram of atheromatous tissue.
  
  
  CONCLUSIONS
  These results indicate that viable previously uncultivable bacterial species are present within atheromas. Our results suggest revisiting the hypothesis that infections may have a causative role in atherosclerotic inflammation and have implications for research regarding novel diagnostics and treatments for cardiovascular disease.
authors.affiliation: Section of Oral and Diagnostic Sciences, College of Dental Medicine, Mailman School of Public Health, Weill Cornell Medical College, New York, NY, USA.
authors.forename: B
authors.initials: B
authors.lastname: Rafferty
authors.forename: D
authors.initials: D
authors.lastname: Jönsson
authors.forename: S
authors.initials: S
authors.lastname: Kalachikov
authors.forename: R T
authors.initials: RT
authors.lastname: Demmer
authors.forename: R
authors.initials: R
authors.lastname: Nowygrod
authors.forename: M S V
authors.initials: MS
authors.lastname: Elkind
authors.forename: H
authors.initials: H
authors.lastname: Bush
authors.forename: E
authors.initials: E
authors.lastname: Kozarov
mesh_terms: D000368:Aged; D017073:Atherectomy; D050197:Atherosclerosis / Q000382:microbiology*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D018920:Coculture Techniques; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000302:isolation & purification; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000382:microbiology; D058226:Plaque, Atherosclerotic / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000302:isolation & purification; D011425:Propionibacterium acnes / Q000302:isolation & purification; D012307:Risk Factors; D013212:Staphylococcus epidermidis / Q000302:isolation & purification; D013291:Streptococcus / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial